The prevailing view of the pathogenesis of the major inflammatory bowel diseases such as Crohns disease (CD) and ulcerative colitis (UC) is that they are caused by dysregulated and therefore enhanced responses to microbial organisms in the gut microbiome that ultimately result in gut inflammation. Perhaps the most important support for this idea is the fact that the various gene polymorphisms associated with these diseases affect the risk for disease development by their effects on mucosal immune homeostasis. In the present study we examined this hypothesis by studies of the abnormalities in mucosal immune function causes by polymorphisms in the LRRK2/MUC19 gene region. This region can be considered a disease hot spot in view of the fact that mutations and single nucleotide polymorphisms (SNPs) in this gene region are risk factors associated with several major diseases including Parkinsons disease, IBD and leprosy. In addition, with respect to IBD this polymorphism has particular significance because the SNP at rs11564258 in the LRRK2/MUC19 locus possesses an odds ratio (O.R.) exceeded only by that of the IL-23R locus among those loci shared by both forms of IBD (CD and UC). To evaluate how the major risk allele located at rs11564258 affects LRRK2 expression we compared LRRK2 mRNA in DCs from patients with CD or LRRK2mRNA and protein levels in lymphoblastoid cell lines (LCLs) from individuals without CD that were heterozygous for the LRRK2 risk allele (G/A) at rs11564258 with control cells without the risk allele (G/G) at this locus. In addition, we compared LRRK2 protein levels in patients with Crohn's disease heterozygous for the polymorphism. The heterozygous cells exhibited significantly increased LRRK2 mRNA obtained from both DCs from patients with CD and LCLs from individuals bearing the polymorphism and increased protein levels in DCs from patients with CD. These important results indicated that the risk allele was associated with increased LRRK2 levels even in individuals without intestinal inflammation and thus differed from previous studies examining this question that was based on studies of another LRRK2 polymorphism marginally associated in patients with Crohn's disease. On the basis of these findings we pursued the study of LRRK2 function in both Lrrk2-transgenic mice (Lrrk2 Tg) with increased levels of LRRK2 expression as well as in Lrrk2 KO mice that lack LRRK2 expression. In initial studies to evaluate global LRRK2 function during colonic inflammation, we subjected the Lrrk2 Tg mice (as well as Lrrk2 KO mice) to DSS colitis. Lrrk2 Tg mice exhibited more severe DSS colitis associated with enhanced pro-inflammatory cytokine secretion as compared with littermate control mice or closely related mice reared in the same environment. In contrast, DSS colitis in Lrrk2 KO mice was slightly less severe than in control mice. To investigate the basis of the increased colitis in LRRK2 Tg mice we examined the capacity of bone marrow-derived dendritic cells from the mice to respond to various innate stimuli and found that, indeed, these cells mounted increased responses to Zymosan-depleted extracts or to fungal antigens that stimulate cells via the Dectin-1 receptor; in contrast, responses to a host of other factors that stimulate cells via TLR receptors did not elicit increased responses. We then conducted a host of studies focused on the effect of LRRK2 expression on the function or expression of the various factors that support NF-kB responses or that result from NF-kB activation and showed quite definitely that LRRK2 enhances NF-kB activation. In related but independent studies we then examined the effects of LRRK2 expression on autophagy induced by M. leprae, organisms that express Dectin-1 stimulating factors and that elicit autophagy in dendritic cells. In extensive studies we showed that increased Lrrk2 expression in the LRRK Tg mice inhibit autophagy and that such inhibition is due to the fact that LRRK2 binds to and induces the degradation of Beclin-1, a key autophagy initiator. Inasmuch as autophagic processes reduce LRRK2 levels, the inhibitory effect of LRRK2 on autophagy could lead to increased LRRK2 levels and greater effects of the latter on NF-kB activation. In a final series of studies that were greatly expanded during this report period and that are most clinically relevant, we showed that a variety of inhibitors of the kinase function of LRRK2 inhibit the capacity of LRRK2 to enhance Dectin-1 responses. In addition, these inhibitors reduce Dectin-1 induction of pro-inflammatory cytokines in Crohn's disease patient cells and inhibit DSS colitis in WT and LRRK2 Tg mice. Finally, these inhibitors neutralized the negative effect of LRRK2 on autophagy. These studies suggest that inhibition of LRRK2 by administration of anti-LRRK2 antibodies have the potential to treat Crohn's disease or ulcerative colitis. In related studies of genetic factors underlying IBD, we now report on a kindred containing two family members (the proband and his aunt) that have identical mutations in one allele of CARD8, a protein that inhibits NLRP3 inflammasome activity and thus have defective CARD8 function that leads to increased NLRP3 activity and increased IL-1b production in the GI tract. CARD8 (Tucan or CARDINAL) is a molecule that binds to NLRP3 and partially inhibits the latter's binding to ASC and CASPASE 1. In the patients studied one allele of CARD8 bears a V44I mutation leading to an amino acid substitution at this site. The proband is a 14 year old boy with refractory GI symptoms suggestive of Crohn's disease whose symptoms subsided only after institution of IL-1b blockers, anakinra or anti-IL1beta neurtralizing antibody. An aunt of this patient with Crohn's also bears this CARD8 mutation, but the mother of this patients is free of Crohn's disease despite bearing this mutation. In the several studies performed to define the abnormality caused by this mutation we showed first that the patients bearing the mutation had increased IL-1beta levels and increased NLRP2 inflammasome responses in dendritic cells stimulated by LPS and nigericin. However, this mutation did not cause abnormalities of NLRC4 Inflammasome. We then showed that the mutated CARD8 exhibits reduced binding to NLRP3 and Caspase I and leads to enhanced ASC polymerization, indicative of enhanced NLRP3 function. Finally, we showed with HEK293 cells that mutated CARD8 acts as a dominant negative in that it reduces the binding to intact CARD8 to NLP3; this explains the fact that individuals bearing this mutation on one allele exhibits increase NLRP3 inflammasome activity. These studies have significance in that they show that abnormal NLRP3 inflammasome activity and Increased IL-1beta production is a possible cause of Crohn's disease and one that will respond to agents that block inflammasome activity. They thus resolve the question raised in studies of murine models as to whether gut inflammation is inflammasome dependent. The answer to this question is that whereas normal inflammasome activity is not in itself an important cause of Crohn's disease, increase inflammasome activity due to a mutation can be a cause;